The development of microarray-based methods in nucleic acid analysis has made rapid progress in recent years. A microarray arrangement is an arrangement of a matrix of probe molecules attached to a sensor in individual, addressable positions in such a way that each position in the array arrangement forms a sensor element by which a target molecule can be detected. The probe molecules used in nucleic acid analysis are usually probe oligonucleotides which have been immobilized (“spotted”) e.g. in an arrangement in the form of a chip in a screen-like matrix on a support. Hybridization with a complementary target nucleic acid results in binding to the probe oligonucleotide. This binding may then be detected by a plurality of alternative methods such that the presence of the target nucleic acid (Z) can be recorded and optionally also quantified owing to the binding event. The detection principle employed may be optical, electrochemical, gravimetric, magnetic and other suitable methods.
Optical methods involve the target nucleic acid or the hybrid of probe oligonucleotide and target nucleic acid being labeled by a label which results in an optically detectable signal. Said label may be a dye, a fluorophore, a chromophore, an intercalating dye, a fluorescent dye or the like. When a binding event occurs, an optically detectable signal can be recorded, for example by a CCD camera capable of taking an image of the entire array, at the addressable position of the particular probe oligonucleotide.
Electrochemical detection methods may involve the probe oligonucleotides being immobilized on an electrochemical sensor. The target nucleic acid or the hybrid of probe oligonucleotide and target nucleic acid is labeled by a label which modifies the electrochemical properties on the sensor element locally at the position of the particular probe oligonucleotide, thus enabling an electric signal, for example a voltage, a current, a change in capacity or the like to be measured. A corresponding method has been disclosed in DE 101 341. This method comprises labeling target nucleic acids with biotin, labeling, after hybridization of the target nucleic acid to the probe oligonucleotide, the bound target nucleic acid with streptavidin-alkaline phosphatase, and presenting to the alkaline phosphatase an enzyme substrate which, when converted by the phosphatase, results in a product which changes conductivity locally, thus enabling a local increase in current to be measured at the electrode on which the probe oligonucleotide has been immobilized.
Gravimetric methods involve recording a signal which results from the change in mass upon hybridization of the target nucleic acid to the probe oligonucleotide. Examples of known methods are “FBAR” and “cantilever” methods.
Magnetic detection involves the probe oligonucleotide being immobilized on a magnetic sensor element, for example a GMR sensor. The target nucleic acid or the hybrid of probe oligonucleotide and target nucleic acid may then be labeled, for example, with paramagnetic particles, for example iron oxide nanoparticles, thus enabling a change in magnetic properties upon binding of the target nucleic acid to be recorded locally at the position of the probe oligonucleotide.
A problem surfacing in all of the detection principles is the fact that qualitative differences between individual sensor elements may appear, which may result in signals of different strengths, especially in the case of highly sensitive and quantitative or semiquantitative measurement methods. Other external influences, for example temperature fluctuations or temperature gradients, flow fluctuations or flow gradients caused by sensor surface fluidics, and other factors, may also result in the fact that not all of the sensor elements in a microarray arrangement have the same sensitivity or, at a correspondingly similar target nucleic acid (Z) concentration, provide a signal having the same strength. Thus, for example, sensor elements on the edge of a microarray arrangement are known to behave differently from sensor elements in the center of a microarray arrangement.
The reliable, error-free operation of assays based on microarrays thus requires a reliable and error-free quality control. The latter must include especially the essential elements of positive and negative control. To this end, a particular sensor element should ideally be calibrated by means of a two-point calibration, i.e. each sensor should be loaded also with two known samples (sample concentrations) in addition to the sample to be determined, with the respective measurement signals being recorded.
This procedure is very difficult to implement, in particular for microarray systems having a high number of sensor elements or addressable positions, since a two-point calibration system usually requires complex and expensive measures. Technology is known to solve or to avoid this problem by designing various sensor element positions in the microarray arrangement as control sensor elements (control spots). The disadvantage here, however, is the necessary assumption that the various sensors behave in an absolutely identical manner, and that the designed control spots are representative of all sensor elements.